Chagas disease caused by Trypanosoma cruzi, is a major public health issue, particularly in the Americas. There are approximately 10 million infected people and more than ten thousand annual deaths from this infection. The available therapeutic agents have limited efficacy beyond the acute phase of the T. cruzi infection, as well as significant side effects, which limit their usefulness. A safe, effective and reliable vaccine would clearly reduce the threat of T. cruzi infections and prevent Chagas disease (chronic infection). However, no suitable vaccine is currently available for protecting against T. cruzi infection, in spite of considerable research in this area. A major hurdle in T. cruzi vaccine development has been the lack of understanding of the requirements for the induction of protective immunity. Recently, we established an effective inducible system for T. cruzi employing the degradation domain based on the Escherichia coli dihydrofolate reductase (ecDHFR). The DHFR degradation domain (DDD) can be stabilized by trimethoprim-lactate and can be used to express detrimental or toxic proteins which can induce a self-destruction process in these parasites. T. cruzi lines with Alpha-toxin, Cecropin A and GFP under the control of DDD with a hemagglutinin tag (HA) were developed. T. cruzi bearing an inducible detrimental/toxin gene allow this parasite to undergo a self- destructive process, resulting in the death of intracellular parasites and the elimination of tissue parasitism. We found that our DDDHA strains were attenuated in mouse experiments producing no pathological changes. Most importantly, inoculation with these DDDHA strains in mice provides significant protection against lethal wild type infection with vaccinated mice producing a high serum level of IFN-? during both immunization and re-infection. CD4+, CD8+ T cells, monocytes and neutrophils undergo robust activation during challenge infection of vaccinated hosts. These transgenic T. cruzi strains, therefore, provide an important new opportunity to understand a protective immune response against T. cruzi infection. Using the GFP-DDDHA strain, we will investigate which adaptive arm of immune system providing the protection. Genetic models (CD8, CD4, B cell knockout mice) and antibody depletions as well as adoptive transfers will be used to investigate which immune cells are important in providing protection in vaccinated mice during re-infection. We will investigate how memory immune cells orchestrate extensive modifications of host innate immune responses in vaccinated host upon re-infection with T. cruzi. Innate immune cells from spleens and livers will be investigated for their activation kinetics by flow cytometry. Both blood phagocytes and NK cells recruitments to the infected spleens will be analyzed by confocal microscopy. CD4+ and CD8+ T cells will be depleted by antibodies and then innate immune cell activation in response to re-infection will be investigated. Global gene expression profile in monocytes upon re-infection will be examined by microarrays. Using bone-marrow chimeras, we will investigate the importance of IFN-? signaling in the innate cells in mediating protection against re-infection. The studies will improve our understanding of how to induce protective responses for vaccine design against T. cruzi infection.